A number of biological processes that are important for normal and pathological states are governed by interactions of cellular proteins mediated by Src homology region 3 (SH3) domains. This disclosure concerns methods and materials for generating SH3 domains with engineered binding properties, and their use as tools in research, therapy, diagnostics and drug discovery.
Virtually all aspects of cellular behavior, such as adaptation of a cell in response to extracellular stimuli by changing of its pattern of gene expression, are regulated and executed by dynamic and ordered proximity of cellular proteins. During the evolution several different types of protein domains specialized in mediating such regulated and specific protein-protein interaction events have emerged. Protein domains of one type typically form large families of homologous but sufficiently divergent members, such that each of them have unique, although often overlapping specificities for ligand binding.
The SH3 domain was first identified as a region of homology among the Src family tyrosine kinases encoded by oncogenic retroviruses and their cellular proto-oncogene counterparts. Thereafter SH3 domains have been noticed in a large number ( greater than 50) of proteins that serve important functions in regulating cell growth, differentiation, and other processes. Because of these functions SH3 domains are intimately involved in pathogenes is of various diseases, in particular cancer. In addition, various microbial pathogens, such as HIV, exploit SH3-mediated processes as a part of their life-cycle. Ability to influence protein complex formation mediated by SH3 domains would therefore have significant therapeutic potential.
SH3 domains are globular protein modules typically consisting of 50-70 amino acids found in many different proteins, particularly proteins involved in cellular signal transduction (Cohen et al. 1995. Cell, 80, 237-248; Dalgarno et al. 1997. Biopolymers, 43, 383-400). SH3 domains mediate inter- and intramolecular interactions by binding to ligands that contain a region with a secondary structure known as the polyproline type II (PPII) helix. These ligands can bind to SH3 domains in two oposite orientations and typically show the xe2x80x9cPxxP motifxe2x80x9d consensus sequences RXxe2x88x85PXXP (SEQ ID NO: 25) and PXxe2x88x85PXP (xe2x88x85 is a hydrophobic amino acid, X is any amino acid) (Feng et al. 1994. Science, 266, 1241-7; Lim et al. 1994. Nature, 372, 375-9). The positioning of the conserved basic residue (usually an arginine, R) in the PxxP motif determines in which orientation the ligand binds its cognate SH3 domain. In addition, there are arypical SH3-ligands with PPII helices that do not conform to such consensus rules. A notable example is the PPII region in Src, which is involved in catalytic autoinhibition by binding to the SH3 domain of Src itself, but contains only one of the two prolines that ordinarily define a PxxP-motif (Xu et al. 1997. Nature, 385, 595-602).
Sequence variation in the PPII helix region involving the consensus as well as the adjacent, non-consensus positions, has been shown to influence the specificity in SH3/ligand complex formation. Examples of preference for targets with atypical PxxP consensus motifs have been provided by studies addressing Abl SH3 ligand selection (Feng et al. 1994. Science, 266, 1241-7; Weng et al. 1995. Mol Cell Biol, 15, 5627-34; Yu at al. 1994. Cell, 76, 933-945), and the CrkN-SH3/C3G peptide complex (Knudsen et al. 1995. EMBO J, 14, 2191-8; Wu at al. 1995. Structure, 3, 216-226). The effect of sequence variation involving the non-consensus residues in the PPII region of SH3-ligands has been best demonstrated by experiments in which distinctive target sequences have been selected for different SH3 domains from libraries of chemically synthesized or phage-displayed random peptides (Sparks et al. 1994. J Biol Chem, 269, 23853-6; Viguera et al. 1994. Biochemistry, 33, 10925-33; Yu at al. 1994. Cell. 76, 933-45). However, despite the above-discussed evidence for specificity, the maximal SH3-binding affinities of short PPII ligand peptides are low, and the relative differences in their binding to different SH3 domains are modest.
By contrast, there is increasing evidence that molecular contacts outside the PPII helix interface can provide significant specificity and strength to SH3-binding. Use of phage-display libraries of longer peptides containing a PxxP motif embedded within random sequence has demonstrated that the flanking residues can increase the selectivity of such ligands, which may show up to 20-to-30 fold differences in their affinities towards different SH3 domains (Rickles et al. 1994. EMBO J, 13, 5598-604; Rickles et al. 1995. Proc Natl Acad Sci U S A, 92, 10909-13, Sparks et al. 1996. Proc Natl Acad Sci U S A, 93, 1540-4). Structural analysis of the interactions of Src-SH3 with two such dodecapeptides revealed that the relatively high specificity and affinity (KD values 0.54 xcexcM and 1.2 xcexcM) of these interactions involved contacts between the flanking residues in the peptides and two loop-like structures in the Src-SH13 domain, which represent regions of high sequence diversity among different SH3 domains and are known as the n-src- and RT-loops (Feng et al. 1995. Proc Natl Acad Sci U S A. 92, 12408-15). Similarly, the sepecific binding of a rationally designed proline-rich ligand to Abl SH3 (KD 0.4 xcexcM for Abl vs. 273 xcexcM for Fyn-SH3) could be explained by corresponding molecular contacts with Abl SH3 (Pisabarro and Serrano, 1906. Biochemistry, 35, 10634-40; Pisabarro et al. 1998. J Mol Biol, 281, 513-521).
Another interaction that has been informative in elucidating the basis of SH3 binding specificity, which also emphasizes the role of the RT-loop, is the complex between HIV-1 Nef and the SH3 domain of the tyrosine kinase Hck. Nef is a 27-34 kD myristoylated protein of primate lentiviruses (HIV-1, -2, and SIVs), and important for development of high viremia and immunodeficiency in the infected host (Harris, 1996. J Gen Virol, 77, 2379-92; Saksela, 1997. Front Biosci, 2, 606-618). Interestingly, Nef has remarkably selective SH3-binding characteristics. It can bind tightly to the Hck-SH3, showing affinity values of approximately KD 0.2 xcexcM as measured by surface plasmon resonance (Lee at al. 1995. EMBO J, 14, 5006-15). In contrast to the strong binding to Hck, Nef has almost a 100-fold lower affinity towards the highly homologous SH3 domain of Fyn. Biochemical and structural studies have revealed that the basis of this selectivity lies in the efficient strategy of Nef for recognition of the non-conserved SH3 residues distinctive to Hck, in particular the side chain of an isoleucine located in the RT-loop of Hck-SH3 (Lee et al. 1996. Cell, 85, 931-942). The region that accommodates the Hck-SH3 RT-loop is composed of multiple non-contiguous parts of the Nef polypeptide, and is located distally from the PPII region in the three-dimensional structure of Nef.
Previous attempts to generate molecules that could compete with naturally occurring SH3-interactions have focused on design or selection from random libraries of peptides and peptide-like molecules that could compete with PPII ligands for their binding to their cognate SH3 domains. Success in such approaches has been reported by a number of groups (see references above). Patent applications for different modifications of this approach have been filed (such as WO 95/24419 and WO 96/03649). However, the relative similarity of the SH3/PPII interface of different SH3/ligand pairs presents a problem far developing highly specific inhibitory molecules. To overcome this problem we have chosen a different approach, which is based on the apparent role of the SH3 domain RT-loop in ligand selection that has been indicated by a number of studies, in particular our previous work on the complex between the HIV Nef protein and the SH3 domain of the cellular Hck tyrosine kinase.
The above observations suggest a general mode where regions in SH3 ligands outside the PPII helix region provide specificity and affinity for binding by contacting regions that are divergent among SH3 domains, in particular residues in the RT-loop. Prompted by this concept, in the present invention we have constructed a large library ( greater than 130 millions) of Hck-derived artificial SH3 domains, in which six non-conserved, Hck-specific residues in the RT-loop have been replaced by a random hexapeptide (termed RRT-SH3 for randomized RT-loop, and expressed these on the surface M13 bacteriophages in order to identify novel SH3 domains with engineered binding characteristics. We show that phage-display is well suited for presentation and selection of modified SH3 domains, and provide strong experimental support for a role of the SH3 RT-loop as a versatile specificity and affinity determinant.
Consequently, as explained hereinbelow, we have found that by randomly manipulating the amino acid sequence comprising the variable region of the RT-loop (in this case six amino acids of the Hck SH3 domain) it is possible to create artificial SH3 domains that bind with unnaturally high affinities and with predetermined binding specificities to different ligand proteins.
The well-characterized interaction between HIV-1 Nef and the SH3 domain of Hck is one of the tightest known SH3-mediated interactions. We have previously shown that a similar capacity for binding to Nef can be transferred to Fyn-SH3 by engineering Hck-like amino acid substitutions into its RT-loop. The present invention is in the finding that, instead of mimicking the structure of a naturally occurring, known cognate SH3 domain, one can generate SH3 domains with desired ligand binding properties by using random manipulation of the RT-loop sequence combined with a powerful affinity or functional selection. Notably the method described in this invention can be used to identify SH3 domains with unnaturally high affinities specific for proteins known to bind to any naturally occurring SH3 domains, as well as to target proteins that are believed to be SH3 ligands but lack an identified SH3 domain-containing cellular partner.
Consequently, the present invention provides a method for generating SH3 domains with tailored binding properties, artificial SH3 domains (termed RRT-SH3 domains) obtained by such a method for use as efficient took in research, diagnostics, therapy and drug discovery.